Assays

ABSTRACT

The present invention is directed to assays that can be used to screen for compounds that act as agonists or antagonists or inverse agonists of bovine adrenal medulla docosapeptide (BAM-22P). The assays are based upon the binding of BAM-22P to the rat and human DRR receptors.

FIELD OF THE INVENTION

The present invention is directed to assay methods that can be used todetermine whether a test compound has activity as a modulator of thebinding and activity of BAM-22P at the rat and human DRRs. Compoundsidentified as being effective modulators have potential use astherapeutic agents in treating pain, neuropathic and inflammatorydisorders.

BACKGROUND OF THE INVENTION

A. Bovine Adrenal Medulla docosapeptide

BAM22 Peptide: YGGFMRRVGRPEWWMDYOKRYG-OH.

The preproenkephalin A (PPA) cDNA was cloned in 1983 (Nature297:431-434, 1982). The PPA gene contains several basic amino acidswithin its coding sequence which can give rise to several peptides uponprotease cleavage. Such peptide cleavage products include BAM12P,BAM20P, BAM22P, MEAGL, MEAP and Peptide E and F (Bovine adrenalmedulla). Some of these peptides have been implicated in neuronalsurvival, and analgesia (Int. J. Devl. Neuroscience 10, No2.pp171-179,1992, Eur. J. Pharmacol., 85,355-356, 1982).

BAM-22P is a peptide precursor of Met-enkephalins (Mizuno, Biochem.Biophys. Res. Commun. 97 (4): 1283-90 (1980). In mammals, BAM-22P hasbeen shown to be present (by immunoreactivity) in peripheral tissue suchas the adrenal medulla, islets of Langerhans (Timiners, Diabetes.; 35(1):52-7 (1986)) and in the central nervous system including manyhypothalamic and thalamic nuclei, inteipeduncular nucleus, substantianigra, the colliculi, periaqueductal gray, parabrachial nuclei,trigeminal motor and spinal nuclei, nucleus raphe magnus and other raphenuclei, nucleus reticularis paragigantocellularis, vestibular nuclei,several noradrenergic cell groups, nucleus tractus solitarius, as wellas in the dorsal horn of the spinal cord (Khachiaturian, J. Comp.Neurol. 220 (3):310-20 (1983).

Although the precise physiological activity of BAM-22P in mammals is notknown, it has been implicated in motor coordination and analgesia (J.Pharmacol Exp Ther 1986 September; 238 (3):1029-1044, Eur. J.Pharmacol., 85.355-356, 1982).

BAM-22P is an orphan peptide and its specific receptor has not beenidentified despite its known ability to bind to the mu, delta and kappaopioid receptors. The present inventors have discovered that BAN/22P isable to activate mammalian dorsal root receptors (DRR), including therat and human DRR receptors.

B. G Protein-Coupled Receptors

G protein coupled receptors (GPCRs) constitute a family of proteinssharing a common structural organization characterized by anextracellular N-terminal end, seven hydrophobic alpha helices putativelyconstituting transmembrane domains and an intracellular C-terminaldomain. GPCRs bind a wide variety of ligands that trigger intracellularsignals through the activation of transducing G proteins (Caron, et al.,Rec. Prog. Horm. Res. 48:277-290 (1993); Freedman, et al., Rec. Prog.Horm. Res. 51:319-353 (1996)).

More than 300 GPCRs have been cloned thus far and it is generallyassumed that there exist well over 1,000 such receptors. Roughly 50-60%of all clinically relevant drugs act by modulating the functions ofvarious GPCRs (Gudermann, et al., J. Mol. Med. 73:51-63 (1995)). Many ofthe clinically relevant receptors are located in the central nervoussystem.

Among the GPCRs that have been identified and cloned is a gene thatencodes a protein known as DRR which is homologous to the receptors ofthe mas oncogene family. A rat counterpart of DRR was found to behomologous and based upon the location of cells expressing DRR mRNA, ithas been proposed that the receptor plays a role in transmission ofpain. However, the endogenous ligand for this family of receptors hasnot previously been identified (Cell: 45, 711-719 1986, JBC273,11867-11873 1998, WO 99/32519).

SUMMARY OF THE INVENTION

The present invention provides assays capable of identifying potentialtherapeutic compounds which are agonists or antagonists of the DRRreceptor. Recombinant cells expressing either rat or human DRR can beused in conjunction with BAM-22P in screening assays designed toidentify agonists and antagonists. Thus, in its first aspect, theinvention is directed to a method of assaying a test compound for itsability to bind to the DRR receptor. This is accomplished by incubatingcells expressing the receptor gene with BAM-22P and test compound. Theextent to which the binding of BAM-22P is displaced is then determined.Radioligand assays or enzyme-linked immunosorbent assays may beperformed in which either BAM-22P or the test compound is detectablylabeled. Although any cell expressing DRR may be used, a recombinantcell expressing a heterologous DRR gene from either the rat or human ispreferred. The term “heterologous” as used herein refers to any DRR genetransfected into a cell, i.e., the term refers to any non-endogenousDRR.

The invention also encompasses methods of determining if a test compoundis an agonist, antagonist, or inverse agonist of BAM-22P binding basedupon a functional assay. One way to carry out such assays is to incubatea cell expressing DRR with the test compound and to then determinewhether intracellular phospholipase C, adenyl cyclase activity orintracellular calcium concentrations are modulated. Results shouldtypically be compared with those obtained when incubations are performedin a similar manner but in the absence of test compound. In general,functional assays of this type will be performed in conjunction withbinding assays of the sort described above. The preferred cell for usein the assays is a recombinant cell that has been transformed with aheterologous DRR gene. Test compounds that act as agonists shouldproduce an increase in phospholipase C, decrease or increase in adenylylcyclase activity or increase in intracellular levels of calcium. Inverseagonists may reduce phospholipase C activity or intracellular calciumlevels, particularly if assays are performed in the presence of a fixedamount of BAM-22P. Antagonists, should block the binding of BAM-22P tothe receptor but not produce the opposite response in terms ofphospholipase C activity or intracellular calcium that is the hallmarkof an inverse agonist.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to assays that can be used to screencompounds for their ability to modulate the binding of BAM-22P to therat and human DRR receptors. Any form of BAM-22P that has been reportedmay be used, but the preferred peptide is 22 amino acids in length andhas the sequence: BAM22 peptide: YGGFMRRVGRPEWWMDYOKRYG-OH. (SEQ IDNO:1). This peptide may be obtained commercially (Bachem) or can besynthesized using standard methodology well known in the art. Thepeptide may be detectably labeled with radioisotopes such as 125, or,alternatively, fluorescent or chemiluminescent labels can beincorporated. Also, the peptide can be joined to enzymes that arereadily detectable such as horseradish peroxidase.

The DRR receptor may be cloned from rat and/or human cells using knownprocesses such as that described in WO 99/32519. The Examples sectionprovides a detailed description of a procedure that may be used incloning DRR. Once obtained, the DRR sequence should be incorporated intoan expression vector with a promoter active in mammalian cells(Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor Press (1989)). Examples of promoters that may be usedinclude that of the mouse metallothionein I gene (Hamer, et al., J. Mol.Appl. Gen. 1:273-288 (1982)); the immediate-early and TK promoter ofherpes virus (Yao, et al., J. Virol. 69:6249-6258 (1995); McKnight, Cell31:355-365 (1982)); the SV 40 early promoter (Benoist, et al., Nature290:304-310 (1981)); and, the CMV promoter (Boshart, et al., Cell41:521-530 (1985)). Vectors may also include enhancers and otherregulatory elements.

Once expression vectors have been constructed, they can be introducedinto a mammalian cell line by methods such as calcium phosphateprecipitation, microinjection, electroporation, liposomal transfer,viral transfer or particle mediated gene transfer. Although othermammalian cells may be used, BEK-293 cells have been found to givesuccessful results and a procedure for expressing DRR in these cells isdescribed in the Examples section. Standard procedures for selectingcells and for assaying them for the expression of DRR (e.g., by Northernanalysis) may be performed.

Once the BAM-22P peptide and cells expressing the rat and human DRRreceptors have been obtained, assays may be performed to determinewhether test compounds have any effect on binding. A wide variety ofdifferent types of assays can be performed using standard methods wellknown in the art. For example, in radioligand binding assays, cellsexpressing DRRs are incubated with BAM-22P and with a compound beingtested for binding activity. The preferred source of DRR isrecombinantly transformed HEK-293 cells. Other cells may also be usedprovided they do not express other proteins that strongly bind BAM-22P.This can easily be determined by performing binding assays on cellstransformed with DRR and comparing the results obtained with thoseobtained using their non-transformed counterparts.

Assays may be performed using either intact cells or with membranesprepared from the cells (see e.g., Wang, et al., Proc. Natl. Acad. Sci.U.S.A. 90:10230-10234 (1993)). As suggested above, the membranes, orcells, are incubated with BAM-22P and with a preparation of the compoundbeing tested. After binding is complete, receptor is separated from thesolution containing ligand and test compound, e.g., by filtration, andthe amount of binding that has occurred is determined. Preferably, theligand used is detectably labeled with a radioisotope such as ¹²⁵I.However, if desired, other types of labels can also be used. Among themost commonly used fluorescent labeling compounds are fluorescein,isothiocynate, rhodamine, phycoerythrin, phycocyanin, allophycocyanino-phthaldehyde and fluorescamine. Useful chemiluminescent compoundsinclude luminol, isoluminol, theromatic of acridinium ester, imidazole,acridinium salt, and oxalate ester.

Nonspecific binding may be determined by carrying out the bindingreaction in the presence of a large excess of unlabeled ligand. Forexample, labeled BAM-22P may be incubated with receptor and testcompound in the presence of a thousandfold excess of unlabeled BAM-92P.Nonspecific binding should be subtracted from total binding, i.e.,binding in the absence of unlabeled ligand, to arrive at the specificbinding for each sample tested. Other steps such as washing, stirring,shaking, filtering and the like may be included in the assays asnecessary. Typically, wash steps are included after the separation ofmembrane-bound ligand from ligand remaining in solution and prior toquantitation of the amount of ligand bound, e.g., by countingradioactive isotope. The specific binding obtained in the presence oftest compound is compared with that obtained in the presence of labeledligand alone to determine the extent to which the test compound hasdisplaced receptor binding.

In performing binding assays, care must be taken to avoid artifactswhich may make it appear that a test compound is interacting withreceptor when, in fact, binding is being inhibited by some othermechanism. For example, the compound being tested should be in a bufferwhich does not itself substantially inhibit the binding of BAM-22P andshould, preferably, be tested at several different concentrations.Preparations of test compound should also be examined for proteolyticactivity and it is desirable that antiproteases be included in assays.Finally, it is highly desirable that compounds identified as displacingthe binding of BAM-22P be reexamined in a concentration range sufficientto perform a Scatchard analysis on the results. This type of analysis iswell known in the art and can be used for determining the affinity of atest compound for receptor (see e.g., Ausubel, et al., Current Protocolsand Molecular Biology, 11.2.1-11.2.19 (1993); Laboratory Techniques inBiochemistry and Molecular Biology, Work, et al., Ed. N.Y. (1978)).Computer programs may be used to help in the analysis of results (e.g.,Munson, P., Methods Enzymol. 92:543-577 (1983)).

Depending upon their effect on the activity of the receptor, agents thatinhibit the binding of BAM-22P to receptor may be either agonists orantagonists. Activation of receptor may be monitored using a number ofdifferent methods. For example, phiospholipase C assays may be performedby growing cells in wells of a microtiter plate and then incubating thewells in the presence or absence of test compound total inositolphosphates (IP) may then be extracted in resin columns, and resuspendedin assay buffer. Assay of IP thus recovered can be carried out using anymethod for determining IP concentration. Typically, phospholipase Cassays will be performed separately from binding assays, but it may alsobe possible to perform binding phospholipase C assays on a singlepreparation of cells.

Activation of receptor may also be determined based upon a measurementof intracellular calcium concentration. For example, transformed HEK-293cells may be grown on glass cover slides to confluence. After rinsing,they may be incubated in the presence of an agent such as Fluo-3, Fluo-4and FURA-2 AM (Molecular Probe F-1221). After rinsing and furtherincubation, calcium displacement may be measured using a photometer.Other types of assays for determining intracellular calciumconcentrations are well known in the ail and may also be employed.

Assays that measure the intrinsic activity of the receptor, such asthose based upon inositol phosphate measurement, may be used in order todetermine the activity of inverse agonists. Unlike antagonists whichblock the activity of agonists but produce no activity on their own,inverse agonists produce a biological response diametrically opposed tothe response produced by an agonist. For example, if an agonist promotedan increase in intracellular calcium, an inverse agonist would decreaseintracellular calcium levels.

The radioligand and cell activation assays discussed above merelyprovide examples of the types of assays that can be used for determiningwhether a particular test compound alters the binding of BAM-22P to thehuman/rat DRR receptors and acts as an agonist or antagonist. There aremany variations on these assays that are compatible with the presentinvention. Such assays may involve the use of labeled antibodies as ameans for detecting BAM-22P that has bound to receptor or may take theform of the fluorescent imaging plate reader assays described in theExamples section herein.

EXAMPLES

I. Methods

Preparation of Clone Rat and Human DRRs:

See description in Patent WO 99/32519

Expression

REK-293 cells were transfected with a mammalian expression constructcoding for the rat and human DRRs (pcDNA 3.0 vector, Invitrogen) usingthe Superfect reagent (Qiagen). A stable receptor pool of DRR wasdeveloped by applying a selection marker (G418, 0.9 mg/ml) and the cellswere maintained in this selection medium. The presence of mRNA specificfor clone DRR was assessed by Northern blot analysis and by the reversetranscriptase polymerase chain reaction (RT-PCR).

Ligands

In order to identify the ligand of clone rat and human DRRs, acollection of peptide and non-peptide ligands was obtained fromcommercial sources (Sigma, CalBiochem, American Peptide Company, Bachem,RBI, Phoenix). The compounds were dissolved in water/DMSO at 3 μM andplaced in 96 well microplates. A total of 1000 compounds (peptides andnon-peptides) were prepared and tested.

Assay

A functional assay was performed with FLIPR (Fluorescent Imaging PlateReader, Molecular Devices) using the fluorescent calcium indicatorFluo-3 (Molecular Probes) on a 96 well platform. BEK-293 cells, eitherexpressing the receptor or wild type cells, were loaded with Fluo-3 asfollows. Stable HEK-293 clones expressing rat and human DRR or parentalcells were plated at a density of 10,000 cells/well in a 96 well plate.On the day of the experiment, the DRR cells were loaded with fluorescentsolution (Dulbecco's modified medium with 10% fetal bovine serumcontaining 4 μM Fluo-3 and 20% pluronic acid). The cells were incubatedat 37 C for one hour in a humidified chamber. Following the incubationstep, cells were washed five times in Hanks' with 20 mM Hepes and 0.1%BSA (pH 7.4). The cells were analyzed using the FLIPR system to measurethe mobilization of intracellular calcium in response to differentcompounds.

II. Results

HEK-293 cells endogenously express some GPCRs such as bradykinin andPACAP receptors which can be used as internal controls for assays. Thebackground signal was established with all of the compounds in theparental HEK-293 cells (non-transfected) using the FLIPR assay. HEK-293cells expressing the clone DRR were stimulated with all compounds andcalcium responses were compared with those in parental BEK-293 cells.Only one compound, bovine adrenal medulla docosapeptide (BAM-22P),consistently elicited signals in the transformed cells but not the wildtype cells. This indicates that BAM-22P is interacting with therecombinantly expressed receptors. Confirmation of this conclusion wasobtained by the observation of a dose-response relationship with BAM-22Pin the cells transfected with DRR, but not in the non-transfected cellsor in cells transfected with other orphan receptors. Thus, it has beenestablished that clone rat and human DRR is a specific receptor forBAM-22P. The rat and human DRR receptors can be used to screen compoundswhich either mimic the action of BAM-22P (agonists) or antagonize theaction of BAM-22P (antagonists).

Screening assays can be performed using the FLIPR assay described above.Alternatively, BAM-22P can be iodinated and used as a tracer inradioligand binding assays on whole cells or membranes. Other assaysthat can be used include the GTPãS assay, adenylyl cyclase assays,assays measuring inositol phosphates, and reporter gene assays (e.g.,those utilizing luciferase, aqueorin, alkaline phosphatase, etc.).

All references cited herein are fully incorporated by reference. Havingnow fully described the invention, it will be understood by those ofskill in the art that the invention may be performed within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

1. A method of assaying a test compound for its ability to bind to amammalian DRR receptor, comprising: a) incubating a cell expressing amammalian DRR receptor with bovine adrenal medulla docosapeptide(BAM-22P), and said test compound; and b) determining the extent towhich the binding of said BAM-22P to said DRR receptor is displaced bysaid test compound.
 2. The method according to claim 1, wherein themammalian DRR is a rat or human DRR.
 3. The method of claim 1 or claim2, wherein said cell expressing DRR is a recombinant cell that has beentransformed with a heterologous DRR gene.
 4. The method of claim 1 orclaim 2, wherein said assay is a radioligand assay and said BAM-22P orsaid test compound is radioactively labeled.
 5. The method of claim 1 orclaim 2, wherein said assay is an enzyme-linked immunosorbent assay(ELISA) and either said BAM-22P or said test compound is joined to anenzyme.
 6. The method of any one of claims 1 to 5, further comprisingdetermining whether said test compound significantly increases ordecreases either the phospholipase C or intracellular calciumconcentration of said cell.
 7. A method of determining if a testcompound is an agonist, antagonist or inverse agonist of BAM-22P,comprising: a) incubating a cell expressing DRR with said test compound;b) determining the intracellular phospholipase C or adenylyl cyclaseactivity or intracellular concentration of calcium of said cell duringthe incubation of step a); c) comparing the results obtained in step b)with the results obtained when incubations are performed in the absenceof said test compound; and d) concluding that said test compound is anagonist of BAM-2P if the level of phospholipase C or adenyl cyclaseactivity or intracellular calcium is significantly higher in thepresence of said test compound than in its absence, or concluding thatsaid test compound is an antagonist of BAM-22P if the level ofphospholipase C or adenyl cyclase activity or intracellular calciumconcentration is significantly lower in the presence of said testcompound than in its absence.
 8. The method of claim 7, wherein saidcell is a recombinant cell that has been transformed with a heterologousDRR gene (rat and human).
 9. The method of either claim 7 or claim 8,wherein said cell expressing DRR and test compound are incubated in amedium further comprising BAM-22P.